FIG. 9.

Altered phosphorylation of VSV M affects interactions with VSV N in cells and virions. (A) Neuroblastomas treated with and without 400 U/mL IFN-β were infected with VSV (MOI = 3), then labeled with ³⁵S-cysteine/methionine (100 μCi/mL). Viral supernatants were collected 10 hpi and cell debris was moved by centrifugation. VSV virions were pelleted via ultracentrifugation (∼302,000 RCF for 15 min), washed with PBS, repelleted, and lysed. The ³⁵S-labeled viral supernatants were then normalized using ³⁵S counts, and resolved via 12% SDS-PAGE. Subsequently the gels were dehydrated in DMSO, infiltrated with 2,5-diphenyloxazole, dried, and exposed to film. Autoradiographs of VSV viral lysates were then used to determine the ratios of virion-associated proteins via densitometric analysis using Un-Scan-It Gel software version 5.1. Statistical analysis revealed a difference between the ratio of virion-associated N and M proteins resulting from IFN-β pretreatment. Due to the nature of N and M interactions necessary for proper virion assembly, the difference in N:M ratio suggested a possible change in the affinity of these proteins for one another. Data represent the mean of three experiments with each experimental condition done in triplicate. Error bars correspond to 95% CI and the p-values were determined by Satterthwaite's method for calculating t-values from independent samples of unequal variances. (B) Immunoprecipitations of cell lysates (at 6 hpi) were conducted using the 23H12 monoclonal anti-VSV M antibody and resolved via 14% SDS-PAGE. Subsequently, the gels were dehydrated in DMSO, infiltrated with 2,5-diphenyloxazole, dried, and exposed to film. VSV M did not co-precipitate detectable N in either treatment group, a result most likely due to a preponderance of M protein either free within the cytosol or in non-RNP complexes. (C) Co-immunoprecipitations as described in B, using anti-VSV N antiserum revealed a change in the ability of N to co-immunoprecipitate M from IFN-β-treated neuronal lysates. Autoradiographs are representative of experiments done in triplicate.